Dr. Aravind helped discover new components in Hedgehog (Hh) signaling pathway. This pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. In collaboration with Dr. Raj Rohatgis group at Stanford Dr. Aravind reported the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac, and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype-the mislocalization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. His evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network. Dr Aravind and his team characterized a novel protein family including mammalian NEMF, Drosophila caliban, yeast Tae2, and bacterial FpbA-like proteins. These are found to be universally distributed across the three domains/superkingdoms of life. Tapping the enormous wealth of genome information that has accumulated since the initial characterization of these proteins, they performed a detailed computational analysis of the family, identifying multiple conserved domains. Domains identified include an enzymatic domain related to the formamidopyrimidine (Fpg), MutM, and Nei/EndoVIII family of DNA glycosylases, a novel, predicted RNA-binding domain, and a domain potentially mediating protein-protein interactions. Through this characterization, they predicted that the DNA glycosylase-like domain catalytically operates on double-stranded RNA, as part of a hitherto unknown base modification mechanism that probably targets rRNAs. At least in archaea, and possibly eukaryotes, this pathway might additionally include the AMMECR1 family of proteins. The predicted RNA-binding domain associated with this family is also observed in distinct architectural contexts in other proteins across phylogenetically diverse prokaryotes. Here it is predicted to play a key role in a new pathway for tRNA 4-thiouridylation along with TusA-like sulfur transfer proteins. Dr. Aravind and his teams study contributed to notable advances regarding the biochemistry and evolution of ADP-ribosylation. They showed that catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems. Dr Aravind and his team investigated the resilience of biochemical activity in protein domains in the face of structural divergence. Dr. Aravind and his group showed that these transformations span a wide spectrum, including simple domains incorporated into larger structural scaffolds, changes in the structural core, major active site shifts, topological rewiring and extensive structural transmogrifications. Proteins from biological conflict systems, such as toxin-antitoxin, restriction-modification, CRISPR/Cas, polymorphic toxin and secondary metabolism systems commonly display such transformations. These include endoDNases, metal-independent RNases, deaminases, ADP ribosyltransferases, immunity proteins, kinases and E1-like enzymes. They further showed that in eukaryotes such transformations are seen in domains involved in chromatin-related peptide recognition and protein/DNA-modification. Intense selective pressures from 'arms-race'-like situations in conflict and macromolecular modification systems could favor drastic structural divergence while preserving function. Dr. Aravind and his team made new contributions towards understanding sexual conflicts in plasmids. Conjugative plasmids are typically locked in intergenomic and sexual conflicts with co-resident rivals, whose translocation they block using fertility inhibition factors (FINs). Dr. Aravind in collaboration with Dr. Arockiasamy at ICGEB described here the first crystal structure of an enigmatic FIN Osa deployed by the proteobacterial plasmid pSa. Osa contains a catalytically active version of the ParB/Sulfiredoxin fold with both ATPase and DNase activity, the latter being regulated by an ATP-dependent switch. Using the Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS), a relative of the conjugative T4SS, They demonstrated that catalytically active Osa blocks T-DNA transfer into plants. With a partially reconstituted T4SS in vitro, they showed that Osa degrades T-DNA in the T-DNA-VirD2 complex before its translocation. Further, they presented evidence for conservation and interplay between ATPase and DNase activities throughout the ParB/Sulfiredoxin fold, using other members of the family, namely P1 ParB and RK2 KorB, which have general functional implications across diverse biological contexts. DDr. Aravind and his team investigated ribosomal biogenesis to identify the elusive nucleases and cofactors involved in the complex rRNA processing events in eukaryotes. Large-scale screens in yeast identified two biochemically uncharacterized proteins, TSR3 and TSR4, as being key players required for rRNA maturation. Using multiple computational approaches Dr. Aravind and his team identified the conserved domains comprising these proteins and establish sequence and structural features providing novel insights regarding their roles. TSR3 is unified with the DTW domain into a novel superfamily of predicted enzymatic domains, with the balance of the available evidence pointing toward an RNase role with the archaeo-eukaryotic TSR3 proteins processing rRNA and the bacterial versions potentially processing tRNA. TSR4, its other eukaryotic homologs PDCD2/rp-8, PDCD2L, Zfrp8, and trus, the predominantly bacterial DUF1963 proteins, and other uncharacterized proteins are unified into a new domain superfamily, which arose from an ancient duplication event of a strand-swapped, dimer-forming all-beta unit. They identified conserved features mediating protein-protein interactions (PPIs) and propose a potential chaperone-like function. While contextual evidence supports a conserved role in ribosome biogenesis for the eukaryotic TSR4-related proteins, there is no evidence for such a role for the bacterial versions.